bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
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Sex-specific changes in gene expression and delayed sex differentiation in response to
estrogen pollution in grayling (Salmonidae)
Oliver M. Selmoni1*, Diane Maitre1*, Julien Roux1,2, Laetitia G. E. Wilkins1, Lucas Marques
da Cunha1, Etienne L. M. Vermeirssen3, Susanne Knörr4, Marc Robinson-Rechavi1,2#, Claus
Wedekind1#
1
Department of Ecology and Evolution, Biophore, University of Lausanne, Lausanne,
Switzerland
2
Swiss Institute of Bioinformatics, Lausanne, Switzerland
3
Swiss Centre for Applied Ecotoxicology Eawag-EPFL, Dübendorf, Switzerland
4
Aquatic Ecology and Toxicology Group, Center of Organismic Studies, University of
Heidelberg, Heidelberg, Germany
* equal contributors
#
shared senior authors
Present addresses:
O. Selmoni: Swiss Federal Institute of Technology (EPFL), Bâtiment GC, 1015 Lausanne,
Switzerland.
L. Wilkins: Department of Environmental Sciences, Policy and Management, 130 Mulford
Hall #3114, University of California, Berkeley, CA 94720, USA
J. Roux: Department of Biomedicine, University of Basel, Hebelstrasse 20, 4031 Basel,
Switzerland
Correspondence: Claus Wedekind, [email protected], orcid.org/0000-0001-6143-4716
Abstract
The synthetic 17α-ethinylestradiol (EE2) is an estrogenic compound of oral contraceptives
and therefore a common pollutant that has been suspected to affect the demography of riverdwelling salmonids. We study a population of European grayling (Thymallus thymallus) that
suffers from sex ratio distortions. Here we test how ecologically relevant concentrations of
EE2 affect sex-specific gene expression around early stages of sex differentiation. We
collected gametes from F1s of wild spawners, used them for in vitro fertilizations, and raised
the resulting embryos singly under experimentally controlled conditions. Embryos were either
exposed to 1ng/L EE2 or sham-exposed. RNA was collected from samples taken 10 days
before hatching, at the day of hatching, and towards the end of the yolk-sac stage, to study
gene expression and relate it to genetic sex (sdY genotype). We found that EE2 affects gene
expression of a very large number of genes especially at the day of hatching. The effects of
EE2 on gene expression is strongly sex-specific. At the day of hatching, EE2 affected about
twice as many genes in females than in males, and towards the end of the yolk-sac larval
stage, EE2 effects were nearly exclusively observed in females. Among the many effects was,
for example, a surprising EE2-induced molecular masculinization in the females’ heads.
Histological examination of gonadal development of EE2-treated or sham-exposed juveniles
during the first 4.5 months after hatching revealed a delaying effect of EE2 on sex
differentiation. Because grayling sex determination goes through an all-male stage (a rare
case of undifferentiated gonochorism), the rate of EE2-induced sex reversal could not be
unequivocally determined during the observational period. However, two EE2-treated genetic
males had ovarian tissues at the end of the study. We conclude that common levels of EE2
pollution affect grayling from very early stages on by interfering with male and female gene
expression around the onset of sex differentiation, by delaying sex differentiation, and by
feminizing some males.
1
bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
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Author contribution
MRR and CW initiated the project. OS, DM, LW, LMC, and CW sampled the adult fish, did
the experimental in vitro fertilizations, and prepared the embryos for experimental rearing in
the laboratory. All further manipulations on the embryos and the larvae were done by OS,
DM, LW, and LMC. The RNA-seq data were analyzed by OS, JR, and MRR, the histological
analyses were done by DM, supervised by SK, the molecular genetic sexing was performed
by OS and DM, and EV supervised the EE2 analytics. OS and CW performed the remaining
statistical analyses and wrote the first version of the manuscript that was then critically
revised by all other authors.
1. Introduction
Endocrine-disrupting chemicals are common pollutants that typically enter the environment
after wastewater treatment. One of the most potent of these micropollutants is the synthetic
17-alpha-ethinylestradiol (EE2) that is used in oral contraceptives and hormone replacement
therapies, and that is more stable and persistent than the natural estrogen it mimics [1]. EE2
concentrations of 1ng/L and higher have been found in river or lake surface waters [2] in lake
sediments [3], and even in groundwater [4].
Exposure to 1 or few ng/L EE2 can be damaging to fish at various developmental
stages. Embryos and early larvae can suffer from increased mortality, reduced growth, or
malformations when exposed to EE2 [5, 6]. In juveniles and adults, exposure to EE2 can
affect the response to infection [7], increase the susceptibility to other micropollutants [8],
generally reduces growth and fertility [8, 9], and can even induce transgenerational effects on
behavior and fertility in F1 [10] and F2 progeny [11]. Studies with experimental populations
kept in 1,100 L ponds revealed population declines at concentrations of 1ng/L EE2 [12], and
long-term, whole-lake experiments revealed significant ecosystem changes after experimental
addition of 5-6 ng/L EE2: local populations of small fish declined (one species nearly got
extinct), average body conditions of other fish, including top predators, declined significantly,
and the prevalence of some zooplankton and insect species seemed to increase [13].
Experimental exposure to EE2, for example of juvenile sticklebacks (Gasterosteus
aculeatus) to 35-40 ng/L, or of juvenile coho salmon (Oncorhynchus kisutch) to 2 or 10 ng/L,
are associated with significant down- and up-regulations of various physiological pathways
[14, 15]. Some of these effects on gene expression may be linked to the toxic effects of EE2
observed in juveniles and adults. However, it is likely that EE2 effects on gene expression
depend on life history and on the developmental stage of an individual, i.e. on the timing of
some physiological pathways in the organism. One important physiological pathway in this
context is sex determination and gonad formation.
Sex determination is probably best seen as a threshold trait, with processes that occur
early in development determining later processes [16]. In amphibians and fishes, these early
processes can be very labile, i.e. potentially modifiable by external factors, even if they often
have a clear genetic basis [17, 18]. Among these external factors that interfere with these
early steps of sex determination are temperature or endocrine disrupting chemicals such as
EE2 [17, 19]. When applied at the beginning of sex determination, they can tip the balance
and cause a sex reversal, i.e. a mismatch between genotypic and phenotypic sex. Such EE2induced sex reversals are sometimes but not always observed. If they are observed, the timing
of exposure seems more relevant than the concentration: Orn et al. [20] found, for example,
nearly complete sex reversal in zebrafish exposure to 5 ng EE2/L during embryo and larval
stages, while exposure to 5-20 ng EE2/L at later stages did not seem to induce sex reversal in
the same species [21].
Apart from gonad development, there can be many other fundamental differences
between male and female development. Males and females may differ, for example, in
average growth, timing of maturation, habitat use, or susceptibility to various stressors
2
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including infections [22]. It is therefore possible that effects of EE2 on the organism crucially
depend on whether or not the organism is exposed to EE2 during the early steps of the sex
determination cascade, when sex reversal is still possible [23].
Exposure to EE2 has been found to induce sex-specific effects in juvenile and adult
fish [23]. These sex-specific effects can be large, as expected by the significant differences in
male and female physiology. It remains unclear whether large differences should also be
expected between the genetic sexes if their gonadal development may have been influenced
by the sex-reversing effects of EE2 [24, 25]. Such questions can be studied if reliable sexlinked genetic markers are available for a given study species.
Here we study the sex-specific effects of ecologically relevant concentrations of EE2
on gene expression and gonad development in grayling (Thymallus thymallus), a riverdwelling salmonid fish that may often be exposed to EE2 pollution. Yano et al. [26]
established sex-linked genetic markers that could be used to determine the genetic sex of
many salmonids, including their sample of grayling taken from a fish farm in France. These
markers could be successfully verified in over 100 phenotypically sexed adult grayling
sampled from our study population [27]. We therefore use them here to separate effects of
EE2 on gene expression in genetic males and females. Maitre et al. [27] found large effects of
genetic sex on gene expression around the time of hatching from eggs, while gene expression
did not seem to differ significantly at late embryogenesis. Their findings thus suggest that the
physiological cascade of sex determination starts during embryogenesis and before hatching.
We therefore study the interaction between EE2 and genetic sex on gene expression in
embryos and larvae. Within-family comparisons are used to minimize potential effects of
genetic variation. Possible interactions between EE2 and genetic sex on gonad development
are studied histologically on samples taken over a period of several months.
2. Methods
2.1 Experimental breeding and raising
Mature males and females were sampled from a captive breeding stock and stripped for their
gametes. These fish are F1 of the natural population described in Wedekind et al. [28]. Their
gametes were used in two full-factorial breeding blocks. For each breeding block, 4 females
were crossed in vitro with 5 males, i.e. 40 (2x4x5) different sibgroups were produced. After
egg hardening for 2 hours, the fertilized eggs were transported to the laboratory where they
were washed and distributed singly to wells of 24-well plates (Falcon, Becton-Dickinson),
following the methods of von Siebenthal et al. [29]. The wells had been filled with 1.8 mL
chemically standardized water [30] that had been oxygenated and temperated before use.
The embryos were incubated at 7°C and left undisturbed until 14 days post
fertilization (dpf), when embryos were exposed either to 1 ng/L EE2 (by adding 0.2 mL water
with a concentration of 10 ng/L EE2, see Brazzola et al. [6] for details), a strain of
Pseudomonas fluorescens (“PF”; 106 bacterial cells per well, the low-virulence strain PF1 in
Clark et al. [31]), simultaneously to EE2 and P. fluorescens (EPF), or sham-treated
(“control”, i.e. only adding 0.2 mL standardized water). A sample of 250 live embryos (EE2treated or sham-treated offspring of one haphazardly chosen female that had been crossed
with five males) was separated for analyses of gene expression, such that we had 25 embryos
for each of the five sibgroups and the two treatments “EE2” and “controls”. The remaining
embryos were raised until 40 dpf, i.e. until several days after hatching, when a subset of each
treatment group was distributed to two 200 L tanks each filled with lake water (pumped from
Lake Geneva at 40 m depth), fed with live Artemia and copepods and later dry food, and
sampled in 5 monthly intervals for histological examination of the gonads (see Maitre et al.
[27] for a description of sex differentiation). For the EE2-treated groups, 200 ng EE2 were
dissolved in 200 L tanks each to reach a starting concentration of 1 ng/L. Every 7 days from
then on, 40 L (i.e. 20%) were replaced with fresh lake water spiked with 40 mL of a 1 µg/L
3
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EE2 stock solution (i.e. 40 L at 1 ng/L EE2). Water samples (100 mL each) were then taken
from each of the 4 EE2-treated tanks 1 hour after this weekly water exchange (T0) and 7 days
later, just before the next water exchange (T7). These water samples were immediately frozen
and stored at -20°C protected from light. Four consecutive T0 and 4 consecutive T7 samples
were each pooled per tank for later determination of EE2 concentrations, i.e. EE2
concentrations were determined for the 4-week intervals these pooled samples covered,
starting 47 dpf, 75 dpf, 103 dpf, and 131 dpf, respectively.
To quantify EE2, the water samples were thawed and filtered over glass fibre filters,
their volume was set to 250 mL and the pH to 3. Four ng/L of EE2 D4 was added as internal
standard and samples were enriched on LiChrolut EN / LiChrolut RP-C18 cartridges that had
been conditioned with hexane, acetone, methanol and finally water (pH 3) [32]. After sample
enrichment, cartridges were dried with nitrogen and eluted with acetone and methanol.
Subsequently, solvents were changed to hexane/acetone 65:35 and extracts were passed over
Chromabond Silica columns [33] and set to a volume of 0.25 mL. LC-MS/MS analysis was
performed on an Agilent6495 Triple Quadrupole. An XBridge BEH C18 XP Column, 2.5 µm,
2.1 mm X 75 mm and an acetonitrile / water gradient was used for liquid chromatography
followed with post-column addition of ammonium fluoride solution. EE2 was quantified by
monitoring the mass transition of 295 to 269, the transition of 295 to 199 served as a qualifier
(internal standard was quantified at the following transitions: 299 to 273 and 299 to 147).
EE2 concentrations in 24-well plates and uptake by embryos are described in Marques
da Cunha et al. (unpublished manuscript). Briefly, plates with newly fertilized embryos were
spiked with EE2 to a concentration of 1 ng/L of EE2 in the plate wells. Analogously, plates
without embryos were spiked to the same concentration. EE2 concentrations in the 24-well
plates with and without embryos were measured in 5 time points across embryo development
(i.e. from 1 dpf until shortly before embryo hatching). While EE2 concentrations have
remained around 1 ng/L in plates without embryos throughout the 5 time points (indicating no
apparent degradation or sorption of EE2 in 24-well plates), in plates with embryos,
concentrations fell to below detection limit (<0.05 ng/L) before the first half of developmental
time (indicating rapid embryo uptake).
In the 200 L tanks, median EE2 concentration were 0.33 ng/L at T0 and 0.11 ng/L at
T7, corresponding to a median reduction of 66% of the EE2 dissolved in water over 7 days
(see Supplementary Figure S1). We found no significant effects of sampling period on the
EE2 measures at T0 (ANOVA, F3 = 1.20, p = 0.35) nor on the weekly reduction of EE2 in the
tanks (F3 = 1.88, p = 0.19; excluding an unexplained outlier, see Figure S1 for discussion).
The median EE2 concentration across T0 and T7 samples in the EE2-treated tanks was 0.2
ng/L from the first water exchange on, i.e. from 47 dpf on (EE2 was also detected in samples
from control aquaria, see Figure S1 for discussion).
For the gene expression analyses, the first sampling of 12 embryos per family and
treatment took place at 21 dpf, i.e. well before hatching could be expected. Embryos were
immediately transferred to RNAlater (Thermo Scientific, Reinach, Switzerland). At 27 and 28
dpf, the incubation temperature was raised to 10°C and 11.5°C, respectively, in order to
induce and synchronize hatching. The second sampling took place at day of peak hatching,
i.e. 31 dpf (8 embryos per family and treatment). The third sampling took place at 52 dpf (5
yolk-sac larvae per family and treatment). Hatchlings and yolk-sac larvae were narcotized
with 0.5mL/L KoiMed (fishmed GmbH, Galmiz, CH) for five minutes and then decapitated.
The heads were immediately transferred to RNAlater. All samples were stored at -80°C.
RNA was extracted using the QIAgen 96 RNeasy Universal Tissue Kit (QIAGEN,
Hombrechtikon, Switzerland). Manufacturer instructions were followed except that
centrifugation (Eppendorf 5804 R centrifuge with an A-2-DWP rotor; Eppendorf,
Schönenbuch, Switzerland) was done twice as long at half the speed. Because the RNA
extraction protocol did not include a DNase treatment, DNA traces inside the RNA samples
4
bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
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were amplified to determine the sdY genotype [26] of each individual, using the 18S gene as
PCR internal control, either in a multiplex reaction used for samples with high amount of
DNA, or after a second PCR amplification in single reactions with half the amounts of the
respective primers each for samples with low DNA content (see Maitre et al. [27] for a more
detailed protocol). Based on the sdY genotype, one female and one male per family and
treatment group was haphazardly chosen for further analyses (in 2 of in total 30 combinations
of family x treatment x time point, two females or two males, respectively, were used because
only one sex could be found in the sample).
The RNA extracts were provided for library preparation in a equimolar concentration
of 6 ng/µL in 100 µL of volume. 50 µL (i.e. 300 ng of RNA) were each used for library
preparation on a robot using the Truseq Stranded RNA protocol (Illumina, Part# 15026495
Rev. A), then introduced in the Illumina sequencing platform (HiSeq 2500) for 100 cycles of
multiplexed paired-end reads sequencing. The total 60 samples were sequenced in ten lanes
(six samples per lane).
2.2 Bioinformatics pipeline
The RNA-seq analysis steps are described in Maitre et al. [27]. To summarize, reads were
quality trimmed or filtered, and PCR duplicates were removed, resulting in a set of 60 RNA
libraries with, on average, 2*40 millions of 80 bp reads each (standard deviation of six
million reads). Reads from 24 libraries were assembled using Trinity (version 2.0.3; [34]).
The resulting assembly was filtered to remove sequences that did not have a significant match
against the UniprotKB/Swiss-Prot database (version of October the 16th 2015) [35] using
blastx (version 2.2.26; [36]). Reads from all libraries were pseudo-mapped onto the
assembled transcriptome using Kallisto (version 0.42) [37], and the read counts of the
different isoforms of each gene were summed up for principal component analysis (PCA) and
differential expression analysis at the gene level. PCA was performed on TMM-normalized
[38] log2(count-per-million) values (CPM). Differential expression analysis was performed
using the limma-voom Bioconductor package (version 3.26.3) [39, 40], with samples quality
weights [41], on CPM values that were additionally cyclic loess normalized. In the linear
model we considered developmental stage, sex and treatment as a combined variable (with
twelve possible levels) and sib-group as an independent variable. A linear model was then fit
for each gene, coefficients and standard errors were computed for all the contrasts of interest.
Q-values [42] were calculated for each gene, and a threshold of q = 0.15 was used to call
differentially expressed genes. Transcripts were annotated by referring to the Gene Ontology
(GO) terms (Biological Function) [43] associated to similar genes in zebrafish. An enrichment
analysis of GO terms was performed on differentially expressed genes using the goseq
Bioconductor package (version 1.22.0; [44])
RNA extraction quality, male sex associated locus PCR, RNA-sequencing reads quality,
transcriptome assembly, gene expression principal component and differential gene
expression within control individuals are shown in Maitre et al. [27].
3. Results
3.1 Differential gene expression
In order to test for sex-specific effects, we compared the changes in gene expression under
EE2 treatment for individuals of the same sex at the same developmental stage (Table 1).
Under EE2 treatment, at the embryo stage there was an altered expression of several hundred
genes in males (Table 1a, Figures S2a, S3, S4, Table S1), but only of a few genes in females
(Table 1a, Figure S2b). At hatching day males showed an alteration in the expression of
nearly 12,000 genes (Table 1b, Figure S2c, S5, S6, Table S2), while females at the same time
point displayed an alteration in the expression of over 24,000 genes (Table 1b, Figure S2d,
5
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S7, S8, Table S3). At the first feeding stage only very few genes appeared altered in males
(Table 1c, Figure S2e), whereas in female still around 14,000 genes were affected (Table 1c,
Figure S2f, S9, S10, Table S4).
In Table 2, the sex-specific alterations in gene expression are split according to the
direction of the changes. Around hatching, about 5,000 genes were up-regulated in EE2treated males while down-regulated in EE2-treated females, and about 4,000 were downregulated in EE2-treated males while up-regulated in EE2-treated females (Table 2). The
remaining sex-specific reactions to the EE2 treatment were mainly up- or down-regulation in
one sex while there was apparently no change in the other sex (Table 2). See Figures S11 and
S12 and Table S5 for EE2 effects on gene expression in both, males and females.
3.2 Does EE2 treatment feminize males and masculinize females?
After focusing on sex-specific gene expression changes induced by EE2 treatment, we
compared control males against EE2-treated females and control females against EE2 treated
males (Table 1). The aim of this analysis was to investigate whether the EE2 treatment would
feminize males, masculinize females, or increase the differences in gene expression between
sexes. At embryo stage, we found three genes differentially expressed between EE2 treated
males and control females (Table 1a) and 863 genes between control males and EE2 treated
females (Table 1a). On hatching day, we found no differences in gene expression levels
between control females and males treated with EE2 (Table 1b) and only two genes differing
between control males and EE2 treated females (Table 1b). At first feeding stage, EE2 treated
males expressed 5,533 genes differently in comparison to control females (Table 1c), while
gene expression in control males differed in 42 genes only from the gene expression of EE2treated females (Table 1c). Overall, there does appear to be transcriptome evidence of
feminization of males and of masculinization of females.
3.3 Gene ontology enrichment analysis
Tables S1-S5 show the top 25 Gene Ontology terms enriched among genes differentially
expressed in the above-described contrasts. Figures S3-S12 illustrate the Gene Ontology
terms significantly enriched, grouped by relatedness. Table 3 shows a summary take-home
from these Gene Ontology enrichments. Interestingly, many differences affect developmental
processes. In male embryos, we also notice response processes, whether endocrine, xenobiotic
or immunological. Finally, in female larvae a wide range of processes are affected, including
behaviour and digestion.
3.4 Sex differentiation
Exposure to EE2 delayed the onset of morphological sex differentiation while exposure to P.
fluorescens (PF) showed no effects (Table 4). Figure 1 shows the rates of juveniles without
any testis or ovarian tissues over the five monthly samples, separately for fish that were or
were not exposed to EE2, to illustrate the delaying effects of EE2. First signs of
morphological sex differentiation could be observed at the 2nd sample, but still more than 30%
of the fish showed no sign of morphological sex differentiation at the end of the observation
period (Figure 1).
Among those fish where first stages of sex differentiation could be identified, only
testis tissue could be observed at the 2nd sampling (79 dpf), while the rate of ovarian tissue
rose quickly to 70.8%, 75.9%, then 79.3% over the 3rd, 4th, and 5th sampling periods,
respectively. The rates of ovarian versus testis tissue did not differ between EE2-treated and
sham-treated grayling (χ2 = 0.26, p = 0.61).
Genetic sexing of all 103 individuals of the 4th and 5th sample (135 dpf and 159-163
dpf) revealed a genetic sex ratio of 55.3% males that did not deviate from equal sex ratio (χ2
= 1.2, d.f. = 1, p = 0.28). Equal sex ratios can therefore be assumed for all earlier samples. At
6
bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
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these two last sampling days, all females except three showed ovarian tissue (ovaries or testisto-ovaries). The three exceptions were from the sham-treated controls and showed testis
tissue, i.e. no genetic female was undifferentiated at that these last sampling dates. In contrast,
45 of a total of 57 genetic males were still undifferentiated at that time, 10 showed testis
tissue, one showed the testis-to-ovaries phenotype, and one had ovaries. The latter two males
with ovarian tissues had both been EE2-treated.
4. Discussion
We tested and described the effects of exposure to low, ecologically relevant, concentrations
of EE2 on sex-specific gene expression in embryos and larvae of grayling, a river-dwelling
salmonid that is often exposed to this type of pollution. From what is known about possible
EE2 effects on fish in general, we expected that this common micropollutant may (i) affect
sex determination of grayling by influencing the few initial triggers that start the canalized
developmental process of gonad formation, and (ii) be toxic to the embryos and larvae
because it interferes with different types of physiological processes, especially those that are
endocrinologically regulated (see references cited in the Introduction). We therefore expected
EE2 to have significant effects on gene expression at various developmental levels, and we
indeed found such effects at all the developmental stages we studied here. However, we had
no clear a priori expectancy about whether EE2 would also affect the genetic males and
genetic females differently at any of these stages.
We started from the premise that sex in gonochoristic species is a threshold trait, i.e. a
canalized developmental process that has one or few initial triggers [16]. In grayling, the
initial trigger (or triggers) that determine phenotypic sex happen during embryogenesis well
before hatching, because over 25,000 genes are already differentially expressed between
genetic males and females at the day of hatching [27]. The 15 genes that Maitre et al. [27]
found to be differentially expressed in genetic males and females at the embryo stage 10 days
before hatching suggest that sex determination starts around then, i.e. at a time when the
embryos had already been exposed to EE2 for several days in the present study.
One possible scenario is hence that EE2 could tip the balance at the early steps of sex
determination so that all individuals follow the developmental process that leads to the female
phenotype regardless of their sdY genotype (i.e. sex reversal of genetic males). If so, EE2
would not be expected to show sex-genotype specific effects on gene expression during later
stages of sex differentiation. However, we found strong interactions between genetic sex and
EE2 on gene expression. These sex-specific reactions to EE2 also depended on the
developmental stages we studied. At embryo stage, expression of only few genes seemed
biased in genetic females, but gene expression in genetic males was already significantly
affected, with about 700 genes up- or down-regulated under the influence of EE2. The
outcome was somewhat reverse at the larval stage: now only few genes of genetic males
seemed to be affected by EE2, while over 14,000 genes were differentially expressed in
genetic females. An even more pronounced effect of EE2 could be seen at the day of
hatching: far over 20,000 genes showed differential expression, and about 9,000 of them were
either upregulated in genetic females and down-regulated in genetic males or down-regulated
in genetic females and upregulated in genetic males.
The strong sex-specific responses to EE2 suggest that exposure to ecologically
relevant concentrations of EE2 during embryogenesis did not simply tip the balance at early
steps of sex determination so that all individuals would become phenotypic females and
would show similar patterns of gene expression from then on. Instead, our observations
suggest that genetic sex largely determined phenotypic sex, and that EE2 then interfered with
sex-specific gene expression, creating the strong sex-specific reactions to EE2. This
conclusion is supported by the observation that the low concentrations of EE2 we used are
commonly observed in natural rivers and streams while there is little evidence for complete
7
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
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and population-wide sex reversal, even if natural populations sometimes show distorted sex
ratios [45].
While the interaction between EE2 and genetic sex on gene expression suggested that
exposure to EE2 is mostly interfering with the development of a phenotype that would
correspond to the genotypic sex, we nevertheless found two genetic males that had developed
ovarian tissues when exposed to EE2. It is possible that we missed some sex-reversal and the
rate of sex reversal is higher, because we learned only during the course of the study that the
grayling is a rare example (and probably even the only one so far) of an undifferentiated
gonochorist that goes through an all-male stage before gonads differentiate into testes and
ovaries [27]. Testis tissue in early juveniles can therefore not be interpreted as evidence for
normal development of a male phenotype, and we probably stopped the study too early to get
a reliable estimate of the rate of sex reversals in grayling exposed to EE2. However, by the
end of the study, nearly all genetic females had developed ovarian tissue. This suggests that
the rate of sex reversals is either low indeed, or that sex reversal would slow down gonad
development so much that we would have missed many sex-reversed individuals within our
observational window. We know of no examples or arguments in the literature that would
support the latter possibility. However, we found that ecologically relevant concentrations of
EE2 could affect sex determination in at least some individuals. It would therefore be
interesting to learn what makes an early embryo susceptible or resistant against chemically
induced sex reversal.
Our gene expression analysis suggested that exposure to EE2 induces effects in the
transcriptomes of the brain that could be interpreted as partial sex reversal, especially around
hatching time when many genes were differentially upregulated in genetic females and
downregulated in genetic male, and vice versa, under the influence of EE2. At the day of
hatching, genetic males appeared to have a somewhat feminized transcriptome when treated
with EE2. This effect seemed to cease before the first feeding stage. Genetic females seemed
to experience some form of masculinization of the transcriptome of the brain, at least from
hatching day to first feeding stage.
Estrogens are known to affect functions of the nervous system, including synapsis
homeostasis [46], neurogenesis [47], and sexual differentiation [47-49]. The latter association
is particularly interesting here because estrogens have repeatedly been shown to induce, to
some degree, a masculinization of the brain [47-49]. This is because androgenic hormones,
produced mainly in the male gonads, are transformed by brain cells into estrogen that
subsequently act locally via specific receptors to induce typical male development [48].
In salmonids, gonadal precursor cells typically differentiate during the weeks that
follow hatching [17, 50]. During this period, the emergence of an endogenous synthesis of
sexual hormones could explain why we observe a divergent response between sexes,
especially if we consider that endocrine active compounds often elicits non-monotonic doseresponses [51, 52]. Such dose-effects could explain why we observed strong sex-specific
responses to EE2 at hatching day and why these responses partly declined towards first
feeding stage. Apart from the likely effects of EE2 on normal development of male and
female phenotypes, exposure to EE2 also affected the expression of genes linked to many
other physiological systems, including, for example, various aspects of the immune system,
the endocrine system, the development of skeletal muscles, or of the digestive system.
In conclusion, exposure to ecologically relevant concentrations of EE2 during early
embryogenesis may potentially tip the balance at early steps of sex determination so that all
individuals follow the developmental process that leads to the female phenotype regardless of
their sdY genotype (i.e. sex reversal of genetic males). If so, and if gene expression is then
mainly determined by this gonad development, EE2 would probably not be expected to show
strong sex-genotype specific effects on gene expression during later stages of sex
differentiation. However, we found strong effects of both, EE2 and genetic sex, on gene
8
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expression at three different developmental stages shortly after the onset of sex determination.
Such sex-specific effects of EE2 suggest that sex reversal has mostly not happened, although
we found two genetic males with gonadal tissues at the end of the study. Our observations
support earlier conclusions that ecologically relevant concentrations of EE2 act mostly toxic
(i.e. affect growth and viability) but have little effect on sex determination in grayling [53],
and that the observed sex ratio distortion may in the population [45] may be due to sexspecific survival instead rather than induced sex reversal.
5. Acknowledgements
We thank B. Bracher, U. Gutmann, C. Küng, R. Mani, M. Schmid, and T. Vuille from the
Fishery Inspectorate Bern for permissions and provision of fish for experimental
fertilizations, the Pachtverein Thun for catching the wild fish, the Saint-Sulpice pump station
for zooplankton supplies, T. Braunbeck for access to the histology laboratory, the Genomic
Technologies Facility of the University of Lausanne for library preparation and sequencing,
and C. Berney, T. Bösch, J. Buser, E. Clark, B. des Monstiers, M. dos Santos, J. Kast, C.
Luca, Y. Marendaz, D. Nusbaumer, D. Olbrich, E. Pereira Alvarez, M. Pompini, A.-L.
Roulin, V. Sentchilo, R. Sermier, B. von Siebenthal, and D. Zeugin for assistance in the field
or the laboratory. This project was authorized by the veterinary authorities of the cantons of
Bern (BE118/14) and Vaud (VD2710; VD2956) and financially supported by the Swiss
Federal Office for the Environment and the Swiss National Science Foundation
(31003A_159579).
6. References
1.
Arnold KE, Brown AR, Ankley GT, Sumpter JP. Medicating the environment:
assessing risks of pharmaceuticals to wildlife and ecosystems. Phil Trans R Soc B
2014; 369(1656):20130569.
2.
Tiedeken EJ, Tahar A, McHugh B, Rowan NJ. Monitoring, sources, receptors, and
control measures for three European Union watch list substances of emerging concern
in receiving waters - A 20 year systematic review. Sci Total Env 2017; 574:11401163.
3.
Yang YY, Cao XH, Zhang MM, Wang J. Occurrence and distribution of endocrinedisrupting compounds in the Honghu Lake and East Dongting Lake along the Central
Yangtze River, China. Environ Sci Pollut Res 2015; 22(22):17644-17652.
4.
Vulliet E, Cren-Olive C. Screening of pharmaceuticals and hormones at the regional
scale, in surface and groundwaters intended to human consumption. Environm Pollut
2011; 159(10):2929-2934.
5.
Morthorst JE, Korsgaard B, Bjerregaard P. Severe malformations of eelpout (Zoarces
viviparus) fry are induced by maternal estrogenic exposure during early
embryogenesis. Mar Environ Res 2016; 113:80-87.
6.
Brazzola G, Chèvre N, Wedekind C. Additive genetic variation for tolerance to
estrogen pollution in natural populations of Alpine whitefish (Coregonus sp.,
Salmonidae). Evol Appl 2014; 7(9):1084-1093.
7.
Rodenas MC, Cabas I, Garcia-Alcazar A, Meseguer J, Mulero V, Garcia-Ayala A.
Selective estrogen receptor modulators differentially alter the immune response of
gilthead seabream juveniles. Fish Shellfish Immun 2016; 52:189-197.
8.
Hua JH, Han J, Wang XF, Guo YY, Zhou BS. The binary mixtures of megestrol
acetate and 17 alpha-ethynylestradiol adversely affect zebrafish reproduction.
Environm Pollut 2016; 213:776-784.
9.
Armstrong BM, Lazorchak JM, Jensen KM, Haring HJ, Smith ME, Flick RW, Bencic
DC, Biales AD. Reproductive effects in fathead minnows (Pimphales promelas)
9
bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
following a 21 d exposure to 17 alpha-ethinylestradiol. Chemosphere 2016; 144:366373.
Volkova K, Caspillo NR, Porseryd T, Hallgren S, Dinnetz P, Porsch-Hallstrom I.
Developmental exposure of zebrafish (Danio rerio) to 17 alpha-ethinylestradiol
affects non-reproductive behavior and fertility as adults, and increases anxiety in
unexposed progeny. Horm Behav 2015; 73:30-38.
Bhandari RK, vom Saal FS, Tillitt DE. Transgenerational effects from early
developmental exposures to bisphenol A or 17 alpha-ethinylestradiol in medaka,
Oryzias latipes. Sci Rep 2015; 5.
Schwindt AR, Winkelman DL. Estimating the effects of 17 alpha-ethinylestradiol on
stochastic population growth rate of fathead minnows: a population synthesis of
empirically derived vital rates. Ecotoxicology 2016; 25(7):1364-1375.
Kidd KA, Paterson MJ, Rennie MD, Podemski CL, Findlay DL, Blanchfield PJ, Liber
K. Direct and indirect responses of a freshwater food web to a potent synthetic
oestrogen. Phil Trans R Soc B 2014; 369(1656).
Prokkola JM, Katsiadaki I, Sebire M, Elphinstone-Davis J, Pausio S, Nikinmaa M,
Leder EH. Microarray analysis of di-n-butyl phthalate and 17 alpha ethinyl-oestradiol
responses in three-spined stickleback testes reveals novel candidate genes for
endocrine disruption. Ecotoxicol Environ Saf 2016; 124:96-104.
Harding LB, Schultz IR, da Silva DAM, Ylitalo GM, Ragsdale D, Harris SI, Bailey S,
Pepich BV, Swanson P. Wastewater treatment plant effluent alters pituitary gland
gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch). Aquat
Toxicol 2016; 178:118-131.
Beukeboom LW, Perrin N: The evolution of sex determination. Oxford: Oxford
University Press; 2014.
Devlin RH, Nagahama Y. Sex determination and sex differentiation in fish: an
overview of genetic, physiological, and environmental influences. Aquaculture 2002;
208(3-4):191-364.
Wedekind C. Demographic and genetic consequences of disturbed sex determination.
Phil Trans R Soc B in press.
Piferrer F. Endocrine sex control strategies for the feminization of teleost fish.
Aquaculture 2001; 197(1-4):229-281.
Orn S, Holbech H, Norrgren L. Sexual disruption in zebrafish (Danio rerio) exposed
to mixtures of 17 alpha-ethinylestradiol and 17 beta-trenbolone. Environ Toxicol
Pharmacol 2016; 41:225-231.
Xu N, Chen PY, Liu L, Zeng YQ, Zhou HX, Li S. Effects of combined exposure to 17
alpha-ethynylestradiol and dibutyl phthalate on the growth and reproduction of adult
male zebrafish (Danio rerio). Ecotoxicol Environ Saf 2014; 107:61-70.
Soffker M, Tyler CR. Endocrine disrupting chemicals and sexual behaviors in fish - a
critical review on effects and possible consequences. Crit Rev Toxicol 2012;
42(8):653-668.
Scholz S, Kluver N. Effects of endocrine disrupters on sexual, gonadal development in
fish. Sex Dev 2009; 3(2-3):136-151.
Senior AM, Lim JN, Nakagawa S. The fitness consequences of environmental sex
reversal in fish: a quantitative review. Biol Rev 2012; 87(4):900-911.
Segner H, Casanova-Nakayama A, Kase R, Tyler CR. Impact of environmental
estrogens on fish considering the diversity of estrogen signaling. Gen Comp
Endocrinol 2013; 191:190-201.
Yano A, Nicol B, Jouanno E, Quillet E, Fostier A, Guyomard R, Guiguen Y. The
sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Ychromosome sequence in many salmonids. Evol Appl 2013; 6(3):486-496.
10
bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
Maitre D, Selmoni OM, Uppal A, Marques da Cunha L, Wilkins LGE, Roux J,
Mobley K, Knörr S, Robinson-Rechavi M, Wedekind C. Sex differentiation in
grayling (Salmonidae) goes through an all-male stage and is delayed in genetic males
who instead grow faster. Submitted manuscript 2017.
Wedekind C, Küng C. Shift of spawning season and effects of climate warming on
developmental stages of a grayling (Salmonidae). Cons Biol 2010; 24(5):1418-1423.
von Siebenthal BA, Jacob A, Wedekind C. Tolerance of whitefish embryos to
Pseudomonas fluorescens linked to genetic and maternal effects, and reduced by
previous exposure. Fish Shellfish Immunol 2009; 26(3):531-535.
OECD guideline for the testing of chemicals 203 (fish acute toxicity test), Annex 2. 9.
[http://www.oecd.org/]
Clark ES, Pompini M, da Cunha LM, Wedekind C. Maternal and paternal
contributions to pathogen resistance dependent on development stage in a whitefish
(Salmonidae). Funct Ecol 2014; 28(3):714-723.
Escher BI, Bramaz N, Quayle P, Rutishauser S, Vermeirssen ELM. Monitoring of the
ecotoxicological hazard potential by polar organic micropollutants in sewage
treatment plants and surface waters using a mode-of-action based test battery. J
Environ Monit 2008; 10:622-631.
Ternes TA, Stumpf M, Mueller J, Haberer K, Wilken R-D, Servos M. Behavior and
occurrence of estrogens in municipal sewage treatment plants -- I. Investigations in
Germany, Canada and Brazil. Sci Total Env 1999; 225:81-90.
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X,
Fan L, Raychowdhury R, Zeng Q et al. Full-length transcriptome assembly from
RNA-Seq data without a reference genome. Nat Biotechnol 2011; 29(7):644--652.
Boeckmann B. The SWISS-PROT protein knowledgebase and its supplement
TrEMBL in 2003. Nucleic Acids Res 2003; 31(1):365--370.
AST® Command Line Applications User Manual.
[https://www.ncbi.nlm.nih.gov/books/NBK279690/]
Bray N, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq
quantification. Nat Biotechnol 2016; 34:525-527.
Robinson MD, Oshlack A. A scaling normalization method for differential expression
analysis of RNA-seq data. Genome Biol 2010; 11(3):R25.
Law CW, Chen Y, Shi W, Smyth GK. voom: Precision weights unlock linear model
analysis tools for RNA-seq read counts. Genome Biol 2014; 15(2):R29.
Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers
differential expression analyses for RNA-sequencing and microarray studies. Nucleic
Acids Res 2015; 43(7):e47--.
Liu R, Holik AZ, Su S, Jansz N, Chen K, Leong HS, Blewitt ME, Asselin-Labat M-L,
Smyth GK, Ritchie ME. Why weight? Modelling sample and observational level
variability improves power in RNA-seq analyses. Nucleic Acids Res 2015; 43(15):e97.
Storey JD. The positive false discovery rate: a Bayesian interpretation and the q-value.
The Annals of Statistics 2003; 31(6):2013--2035.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP,
Dolinski K, Dwight SS, Eppig JT et al. Gene Ontology: tool for the unification of
biology. Nat Genet 2000; 25(1):25-29.
Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNAseq: accounting for selection bias. Genome Biol 2010; 11(2).
Wedekind C, Evanno G, Székely T, Pompini M, Darbellay O, Guthruf J. Persistent
unequal sex ratio in a population of grayling (Salmonidae) and possible role of
temperature increase. Cons Biol 2013; 27(1):229-234.
11
bioRxiv preprint first posted online May. 9, 2017; doi: http://dx.doi.org/10.1101/135210. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
46.
47.
48.
49.
50.
51.
52.
53.
Saldanha CJ, Remage-Healey L, Schlinger BA. Synaptocrine signaling: steroid
synthesis and action at the synapse. Endocrine Reviews 2011; 32(4):532-549.
Diotel N, Le Page Y, Mouriec K, Tong SK, Pellegrini E, Valliant C, Anglade I, Brion
F, Pakdel F, Chung BC et al. Aromatase in the brain of teleost fish: Expression,
regulation and putative functions. Front Neuroendocrinol 2010; 31(2):172-192.
Wu MV, Manoli DS, Fraser EJ, Coats JK, Tollkuhn J, Honda SI, Harada N, Shah NM.
Estrogen masculinizes neural pathways and sex-specific behaviors. Cell 2009;
139(1):61-72.
Wu MV, Shah NM. Control of masculinization of the brain and behavior. Current
Opinion in Neurobiology 2011; 21(1):116-123.
Krisfalusi M, Cloud JG. Gonadal sex reversal in triploid rainbow trout (Oncorhynchus
mykiss). Journal of Experimental Zoology 1999; 284(4):466-472.
Li L, Andersen ME, Heber S, Zhang Q. Non-monotonic dose-response relationship in
steroid hormone receptor-mediated gene expression. Journal of Molecular
Endocrinology 2007; 38(5-6):569-585.
Vandenberg LN, Colborn T, Hayes TB, Heindel JJ, Jacobs DR, Lee DH, Shioda T,
Soto AM, vom Saal FS, Welshons WV et al. Hormones and endocrine-disrupting
chemicals: low-dose effects and nonmonotonic dose responses. Endocrine Reviews
2012; 33(3):378-455.
Pompini M, Buser AM, Thali MR, Von Siebenthal BA, Nusslé S, Guduff S,
Wedekind C. Temperature-induced sex reversal is not responsible for sex-ratio
distortions in grayling Thymallus thymallus or brown trout Salmo trutta. J Fish Biol
2013; 83(2):404-411.
12
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
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Table 1. Number of genes that are differentially expressed (q<0.15) in males and females
tested at (a) embryo stage, (b) hatchling stage, and (c) larval stage at the onset of exogenous
feeding, each after they had been exposed to EE2 or sham exposed.
shamexposed
females
EE2exposed
males
EE2exposed
females
Sham-exposed males
Sham-exposed females
151
704
3
863
32
Sham-exposed males
Sham-exposed females
25,3721
11,619
0
2
24,728
Sham-exposed males
Sham-exposed females
1
reported in Maitre et al. [27]
1,1101
4
5,533
41
14,395
a) Embryos
b) Hatchlings
c) Larvae
590
591
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
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Table 2. Number of genes that were upregulated, i.e. had a positive log fold change of
expression with q > 0.15 (UP), experienced no significant change in expression (NO), or were
downregulated (DO) under exposure to EE2.
.
Males UP
Males NO
Males DO
Total
Females UP
Females NO
Females DO
Total
0
245
1
246
12
34,613
18
34,643
0
457
1
458
12
35,315
20
35,347
Females UP
Females NO
Females DO
Total
147
1,341
4,962
6,450
7,959
8,267
7,495
23,721
4,043
1,009
117
5,169
12,149
10,617
12,574
35,340
Females UP
Females NO
Females DO
Total
3
1
0
4
8,589
20,949
5,803
35,341
0
0
0
0
8,592
20,950
5,803
35,345
a) Embryos
b) Hatchlings
c) Larvae
598
599
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Table 3. Summary interpretation of the differential gene expression analysis. The
characterization of the biological processes relies on the gene ontology enrichment analysis of
differentially expressed genes. Feminization and masculinization represent the situation where
few genes (<100) are detected as differentially expressed, under EE2 treatment, in
comparison to control female or control male, respectively. See Supplementary Figures S2S13 and Tables S1-S5 for more detailed information.
1
Developmental
stage
Sex
differences
in gene
expression 1
Embryos
Weak
Hatchlings
Strong
Larvae
Strong
EE2-effects in males
On genes associated with central
nervous system development,
immune response development,
endocrine system and response to
xenobiotic stimuli.
On genes associated with skeletal
muscle function and locomotion,
metabolism of fatty acids and
carbohydrates, DNA-repair and
hindbrain morphogenesis.
Possible feminization.
A few genes only.
EE2- effects in
females
Unclear.
On genes associated with
immune system activation,
DNA-repair, axonal growth and
synaptic transmission. Possible
masculinization.
On genes associated with DNArepair, signal transduction,
immune system activation,
carbohydrate metabolism,
mechanosensory behavior and
digestion. Possible
masculinization.
Results from Maitre et al. [27]
15
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Table 4. Likelihood ratio test on rate of undifferentiated gonads explained by time point of
sampling and exposure to ethinylestradiol (EE2), the bacterium P. fluorescens (PF), or both
(EE2 x PF). Ntotal = 251.
Effect
Time
EE2
PF
Time x EE2
Time x PF
EE2 x PF
Time EE2 x PF
χ2
72.3
<0.1
<0.1
9.5
0.9
<0.1
4.7
d.f.
4
1
1
4
4
1
4
P
<0.0001
1.0
1.0
0.05
0.92
1.0
0.32
615
616
617
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620
621
622
623
Figure 1
Rates of undifferentiated males and females (i.e. no testis nor ovarian tissues) when exposed
to EE2 (closed symbols) or sham exposed (controls, open symbols), with or without
additional exposure to P. fluorescens during embryogenesis each (exposure to the microbe
showed no significant effects on sex differentiation). See text and Table 4 for statistics.
624
17